The Relationship of Circulating Choline and Choline-Related Metabolite Levels with Health Outcomes: A Scoping Review of Genome-Wide Association Studies and Mendelian Randomization Studies

Choline is essential for proper liver, muscle, brain, lipid metabolism, cellular membrane composition, and repair. Understanding genetic determinants of circulating choline metabolites can help identify new determinants of choline metabolism, requirements, and their link to disease endpoints. We conducted a scoping review to identify studies assessing the association of genetic polymorphisms on circulating choline and choline-related metabolite concentrations and subsequent associations with health outcomes. This study follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement scoping review extension. Literature was searched to September 28, 2022, in 4 databases: Embase, MEDLINE, Web of Science, and the Biological Science Index. Studies of any duration in humans were considered. Any genome-wide association study (GWAS) investigating genetic variant associations with circulating choline and/or choline-related metabolites and any Mendelian randomization (MR) study investigating the association of genetically predicted circulating choline and/or choline-related metabolites with any health outcome were considered. Qualitative evidence is presented in summary tables. From 1248 total reviewed articles, 53 were included (GWAS = 27; MR = 26). Forty-two circulating choline-related metabolites were tested in association with genetic variants in GWAS studies, primarily trimethylamine N-oxide, betaine, sphingomyelins, lysophosphatidylcholines, and phosphatidylcholines. MR studies investigated associations between 52 total unique choline metabolites and 66 unique health outcomes. Of these, 47 significant associations were reported between 16 metabolites (primarily choline, lysophosphatidylcholines, phosphatidylcholines, betaine, and sphingomyelins) and 27 health outcomes including cancer, cardiovascular, metabolic, bone, and brain-related outcomes. Some articles reported significant associations between multiple choline types and the same health outcome. Genetically predicted circulating choline and choline-related metabolite concentrations are associated with a wide variety of health outcomes. Further research is needed to assess how genetic variability influences choline metabolism and whether individuals with lower genetically predicted circulating choline and choline-related metabolite concentrations would benefit from a dietary intervention or supplementation.


Introduction
Choline is an essential nutrient with defined Dietary Reference Intake values in the United States and Canada since 1998 [1].It is essential for proper liver, muscle, and brain functions, lipid metabolism, and cellular membrane composition and repair [1][2][3][4].Choline depletion/repletion studies have linked choline deficiency with liver dysfunction and muscle damage [5,6].Choline intake and adequate fetal choline supply are particularly critical during development, influencing the risk of neural tube defects [7][8][9], modifying septo-hippocampal circuitry and brain function [10], as well as postnatal cognition and chronic disease risk factors [11].Higher choline intake in adults has been linked to better cognitive performance and brain morphology [12].Data from the NHANES show estimated choline intakes to be slightly above 300 mg/d for nonpregnant, nonlactating individuals [13][14][15].A large percentage of individuals are not consuming the Adequate Intake (AI) for choline, with a mere 10% of Americans and 8% of pregnant women currently meeting their gender-and life-stage specific AI for choline [13].Recently, researchers highlighted the difficulty of meeting the AI for choline without egg intake or supplementation [13].Choline in the diet is absorbed and can be partitioned postconsumption toward several different metabolic fates and is present in circulation as free choline or as metabolites [e.g., betaine, phosphatidylcholines (PCs), sphingomyelins (SMs), and acetylcholine); unabsorbed choline can be scavenged by the microbiota and metabolized to trimethylamine (TMA), which is absorbed and oxidized to TMA N-oxide (TMAO).Circulating choline-related metabolite concentrations are the most readily accessible for clinical assessment but are often challenging to interpret, as they represent an aggregate of dietary inputs, endogenous synthesis, and various concentrations of homeostatic regulation across absorption, tissue-level distribution, metabolism, and excretion.
Although these numerous inputs add complexity to the interpretation of circulating choline-related metabolites, such complexity lends itself to discovery.Genetic variants associated with circulating choline-related metabolites identified in Genome-Wide Association Studies (GWAS) have the potential to identify novel determinants of choline's metabolic handling as well as potential candidates for gene-x-diet interactions (i.e., nutrigenetics).GWAS facilitates the identification of novel regulators of whole-body and cellular choline metabolism beyond the extensive literature of candidate gene approaches interrogating common variants throughout one-carbon metabolism on choline requirements and metabolism [16].Recently, the capacity to assess genetic determinants of circulating metabolites has become possible because of the increase in large cohort studies with both high-throughput DNA sequencing data as well as untargeted metabolomic approaches.However, a targeted examination of these analyses' results as they relate specifically to choline has not been undertaken.
Understanding genetic determinants of circulating cholinerelated metabolites is not only beneficial for identifying new determinants of choline metabolism and requirements but also for linking choline metabolism to disease endpoints.Indeed, such variants may be used in instrumental variable (IV) analyses [i.e., Mendelian Randomization (MR)] to identify possible links between circulating choline-related metabolites and disease endpoints [17][18][19].Although these links do not immediately infer causality, they may provide more robust and reliable associations, as they serve as proxies of lifetime exposure and are less prone to confounding and reverse causation when the appropriate IV is used [20].However, until recently, the lack of genetic variants associated with circulating choline-related metabolites has hampered the use of MR to link choline metabolism to potential disease endpoints.
Thus, we have undertaken a scoping review of genetic epidemiological studies, namely GWAS and MR approaches, that have captured genetic data or utilized genetic IVs, measured circulating choline and choline-related metabolites, and examined associations with any health outcomes.The results of this review summarize the current literature regarding metabolic-xgenetic interactions that influence choline metabolism and the relationship of circulating choline to health outcomes.

Methods
A scoping review was conducted and followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement scoping review extension [21,22].The PICO (population, interest, context, and outcome) framework was used to search and identify relevant literature [23,24].

Literature search and selection
Literature was searched to September 28, 2022, in 4 databases: Embase, Ovid MEDLINE, Web of Science (all databases), and the Biological Science Index.Searches included key terms related to circulating choline and choline-related metabolites, as well as GWAS and MR study designs.Results were limited to human studies of any duration.The complete search strategy for MEDLINE is presented in the supplement.The database search was supplemented with 3 additional searches for reports published up to December 2022.First, articles were hand-selected for screening based on expert knowledge.Second, references were mined from articles suspected of reporting on geneticrelated choline/choline metabolite data in supplemental files.Third, the National Human Genome Research Institute-European Bioinformatics GWAS Catalogue [25] was searched for choline-related studies and single nucleotide polymorphisms.The supplemental files of these articles were double-screened and selected for inclusion based on the screening criteria and selection process.Any GWAS investigating genetic variant associations with circulating choline and/or choline/related metabolites and any MR study investigating the effect of circulating choline and/or choline-related metabolites on any health outcome were considered.
Duplicate articles were removed in EndNote (Clarivate Analytics) version X9 before screening.Titles and abstracts were screened by 2 independent investigators using the Rayyan web (Rayyan Systems, Inc.) application for systematic reviews [26].Full-text documents were screened by 1 investigator, and exclusions were double-screened by a second independent investigator.Conflicts were resolved by discussion between the 2 investigators.The complete eligibility criteria used during screening are presented in Table 1.

Data extraction
Data were extracted using an internally created form.Data fields include author, year, title, funding source, primary study aim, study design, cohort/data source, cohort ethnicity, sample size, cohort age, percent female, relatedness, choline/choline metabolite type, and health outcome(s) assessed.Quantitative results were separately extracted only for MR studies and only if the result was statistically significant according to the studyreported significance threshold.Extracted results included the MR method, exposure, outcome, effect size, 95% confidence interval, and P value.

Data reporting and synthesis
Study characteristics and qualitative data are presented in separate tables for GWAS and MR studies to facilitate narrative synthesis.A summary of health outcomes found to be significantly associated with choline or choline-related metabolites across included studies was reported in tables, and a heatmap was used to highlight common findings.Summary data were calculated using Microsoft Excel version 16.70.

Results
Out of 1248 total reviewed articles, 53 met the inclusion criteria (GWAS ¼ 27; MR ¼ 26). Figure 1 outlines the study search and selection process.A list of excluded full-text articles and reasons for exclusion is presented in Supplementary Table 1.The results for GWAS and MR studies are presented in separate sections below.

GWAS
The 27 included GWAS studies utilized data from 64 total data sources representing 33 unique cohorts.The majority of these cohorts included Europeans (76%), whereas the others represented transethnic populations (12%), African Americans (6%), and individuals from Australia, Qatar, or South Korea (1 cohort each).Of the unique cohorts, 58% included unrelated individuals, 39% included related individuals (e.g., families, siblings, twins, or spouses), and for 1 cohort, relatedness was not clear.In 4 of the GWAS studies, related individuals were excluded, or relatedness was otherwise accounted for in the analyses.The reported mean age of participants was 20 y or greater for all data sources; the highest reported participant age was 81 y.Age was not reported for 10 data sources from 7 of the included studies.In the 64 reported data sources, the study population size ranged from 91 to 898,130 participants, where 34% of reported data sources had fewer than 1000 participants, 50% of data sources had 1000 to 5000 participants, and the remaining 16% of data sources had >5000 participants.
From the 27 included GWAS articles, 42 unique circulating choline metabolites were tested in association with genetic variants [primarily TMAO, betaine, SMs, lysophosphatidylcholines (LysoPCs), and PCs].A full list of choline metabolites and key findings reported in the individual GWAS are presented in Table 2 .
5000-100,000 participants, and the remaining 16% of data sources had >100,000 participants.Of the unique cohorts, 34% included unrelated individuals, 25% included related individuals (e.g., families, siblings, twins, or spouses), 6% included combination relatedness (used >1 cohort with different relatedness status), 10% of studies did not report relatedness, and for 25%, relatedness was not clear.MR studies investigated associations between 52 total unique choline metabolites and 66 unique health outcomes.Many included articles reported significant associations between multiple choline types and multiple health outcomes.Of these, 47 significant associations were reported between 16 metabolites (primarily choline, LysoPCs, PCs, betaine, and SMs) and 27 health outcomes.A complete summary of significant MR results is displayed in  3.
Regarding the current state of the literature, the following health outcome categories have been explored: cardiovascular, metabolic, brain and behavioral, kidney, bone, cancer, eye, lung, and gastrointestinal.Cardiovascular and metabolic health outcomes have been most frequently studied in relation to genetically predicted circulating choline/choline metabolites, with 9 cardiovascular [60,64,73] and 11 metabolic-related outcomes [18,19,55,60,66,68,69,73] reported to have significant associations.Metabolic-related outcomes include BMI, dyslipidemia, fasting plasma glucose, HDL cholesterol, HOMA-IR, insulin, overweight/obesity, percent body fat, type II diabetes (T2D), triglycerides, and waist-to-height adjusted BMI.Only 1 study explored the relationship with gastrointestinal health, and the results were not significant [76].Four MR articles investigated genetically predicted choline/choline metabolites and kidney health outcomes, all reporting significant associations with various metabolites [60,64,65,69].Two MR articles investigated bone health, and 1 study reported significant associations with PCs [58,77].Lastly, 2 MR articles investigated cancer health outcomes, with 1 significant association between SM and estrogen receptor-positive breast cancer-specific survival [54,59].

Discussion
The results of our scoping review detail numerous genetic loci associated with circulating choline and choline-related metabolites and their relationships to various health outcomes.These findings represent a rich dataset to be explored both in further genetic epidemiological investigations as well as experimental approaches, including 1) cell-based functional analysis to determine the relevance of hits to choline handling across subcellular compartments; 2) concordance of identified loci with genomic screens under conditions of varied choline nutriture; 3) targeted analysis of the choline metabolome and choline handling (via isotope tracing) in transgenic animal models with whole-body and/or tissue-specific loss of identified loci; 4) ADHD, Attention-deficit/hyperactivity disorder; BMI, body mass index; ERþ, estrogen receptor positive; HDL cholesterol, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance; LysoPC, lysophosphatidylcholine; PC, phosphatidylcholine; RR, relative risk; SM, sphingomyelin; T2D, type II diabetes; TG, triglyceride; TMAO, trimethylamine N-oxide; 1-arachidonoyl-GPC, 1-arachidonoyl-glycerophosphocholine; 1-docosahexaenoyl-GPC, 1-docosahexaenoyl-glycerophosphocholine.
targeted analysis of the choline metabolome in humans across varied genotypes and genosets of identified loci under different choline intake conditions.A recent publication demonstrated the utility of utilizing GWAS hits of circulating choline and performing functional analyses of such hits to identify novel choline transporters, validating the utility of this dataset [78].
Notably, although this current investigation highlights the utility of examining existing genetic variant-metabolite relationships for identifying novel routes of investigation, this evidence base remains in its infancy.The studies and associations identified in this literature base do not represent cause and effect; however, they do identify areas for further research.Heatmap of significant associations reported in Mendelian randomization studies.The scale and colors depict the number of articles reporting significant associations between a circulating choline-related metabolite and a health outcome. 1 GPC, LysoPC, and SM include multiple metabolite forms: GPC (1-arachidonoyl-GPC, 1-docosahexaenoyl-GPC), LysoPC (total LysoPC, LysoPC 14:0/0:0, LysoPC 15:0, LysoPC 16:1), and SM (total SM, SM C34:0, SM C36:0, SM C34:

GWAS results
GWAS included in this scoping review report many genetic polymorphisms that are significantly associated with circulating choline and choline-related metabolite concentrations, revealing loci that influence the homeostatic processes maintaining circulating concentrations.As expected, given the diversity of metabolites captured in our search, the candidate loci that emerged reflect the diversity of physiological processes that choline-related metabolites intersect with, as well as the different analytic strategies (e.g., adjusting for blood lipid level when assessing genetic determinants of circulating lipid-soluble choline metabolites).Some identified loci are unsurprising and represent relatively direct metabolically proximal genes (fatty acid desaturase 1 (FADS1) association with PCs in 3 studies [31,40,44]), whereas other variants have less obvious links to circulating metabolites and may reveal novel distally related metabolic networks (spectrin repeat containing envelope protein 2 (SYNE2) association with SMs in 3 studies [40,44,48]).The key findings from our search extend other work reporting on single nucleotide polymorphisms associated with dietary choline deficiency [16], reporting on several genetic variant associations with lower circulating choline concentrations [27,38,43,47].An important consideration is that the path from GWAS to biology is not straightforward because many loci contribute to standing genetic variation.Furthermore, single nucleotide polymorphisms shown to exert effects on metabolic pathways typically only contribute a small amount to metabolic insufficiency, which is often difficult to distinguish from background variation [79].Studies exploring genetic risk scores with choline-related polymorphisms could help determine combined genetic risk and reveal a greater proportion of the variation in circulating choline-related metabolites that can be explained by genetics.

MR studies results
Many metabolic health outcomes related to circulating choline metabolites with significant associations have been investigated across multiple populations.Common relationships assessed included BMI (in kg/m 2 ), waist-to-height adjusted BMI, blood lipid concentrations, glucose-insulin dynamics, body composition (e.g., percent body fat), and T2D.LysoPCs were most associated with metabolic health outcomes, with betaine, choline, PCs, SMs, and TMAO also showing significant associations.Across MR studies, significant associations were most frequently reported for T2D, with genetically associated circulating betaine, choline, LysoPCs, PCs, SMs, and TMAO being the most widely investigated IVs.The risk of T2D varied between different choline and choline-containing metabolite concentrations, even within the same class of metabolites.For example, T2D risk was greater with higher genetically predicted serum choline [60], TMAO [64], and SM C34:1 [72].However, in other studies, T2D risk was lower given higher genetically predicted serum choline and PC (mediated by sodium-glucose cotransporter 2 (SGLT2) inhibition), LysoPC, and SM C34:0 and C36:0 [19].This phenomenon may highlight how the MR technique relies on all the assumptions necessary for unbiased IV analysis, including the requirement that there be no path from genotype to outcome except through the exposure of interest (i.e., the genotype can neither be confounded nor can it induce the outcome via other pathways [20]).The largest number of significant associations reported for a cardiovascular disease outcome was systolic blood pressure (SBP), showing relationships with LysoPCs [69], TMAO [18,64], SM (OH) C32:2 [73], and SM (2OH) C30:2 [73].Additionally, the direction of the association with a health outcome varied by metabolite.For example, genetically predicted TMAO [64] and LysoPCs [69] showed positive relationships with SBP, whereas SMs showed negative relationships with SBP [73].As previously mentioned, 1 study investigating choline deficiency during pregnancy showed an increased risk of hypertension in rats [11].However, the studies from this review did not identify an association between circulating choline concentrations and hypertension in adults.Although studies have shown that choline deficiency leads to liver dysfunction and muscle damage [6,11], no MR articles investigating these health outcomes were identified in this review.
Fewer articles investigating brain and behavioral outcomes were present in the peer-reviewed literature.Previous research has suggested a beneficial impact of higher dietary choline intake on brain/neurodevelopment [80,81], schizophrenia risk [10], and cognitive decline, as evidenced by differences in brain white matter hyperintensity volume [12].We identified 4 brain and behavioral outcomes within MR studies that were shown to be associated with a genetically predicted circulating choline metabolite [Attention-deficit/hyperactivity disorder (glycerophosphocholine (GPC)) [71]; bipolar disorder (GPC) [61]; Alzheimer's disease (SM) [67]; Anorexia Nervosa (SM) [61]].In contrast, Alzheimer's disease was not significantly associated with choline, betaine, or TMAO in Zhuang et al., 2021 [75], suggesting polymorphisms in SM production may be more critical to Alzheimer's outcomes.Only 1 study reported on associations with genetically predicted GPC/betaine and major depression, autism spectrum disorder, and schizophrenia health outcomes, and associations were nonsignificant [71].Considering the role of choline in sphingomyelin production and brain health, these outcomes may warrant further investigation.

Strengths and limitations
This scoping review is limited in several ways.Individual genetic variants can have varying influences on health outcomes, having main, partial, or no effect at all.The existing GWAS literature examining genetic determinants of circulating metabolites remains in its infancy and is limited by predominantly semi-quantitative assessments of choline and its related metabolites, as well as limited capacity to identify specific molecular species (especially among the diverse lipid metabolites varying in their fatty acid composition).Improvement in identification and quantitation from untargeted approaches, as well as funding and interest for targeted analyses in large cohorts, will likely address these concerns in the future.Generally, across the GWAS-metabolomic literature, there is a great need to standardize approaches regarding sample matrices and collection strategies, quantitation of metabolites, statistical approaches (including covariate adjustment), and attempts to incorporate nutrient intake and nutritional status indicators into analyses.Indeed, the metabolome is strongly influenced by dietary intakes genetic variants can influence diet intakes, and dietary intakes can reveal or obscure metabolite-disease relationships, such as in the case of the methylenetetrahydrofolate reductase (MTHFR) C477T variant, its interaction with dietary folate/folic acid intake, and the risk of elevated homocysteine and cardiovascular disease.
With respect to MR analyses, it is likely that several of the existing genetic variants associated with choline metabolites are subject to the well-recognized limitations of MR, including weak instrument bias, pleiotropy, and confounding [20].Confounding is of particular concern in the case of choline and choline-related metabolites, as circulating concentrations, especially unfasted samples, are sensitive to intake, and food sources of choline and its related metabolites are found across several food groups with intakes of additional nutrients/bioactive compounds that may influence disease relationships (e.g., animal-source foods for choline; grains and leafy greens for betaine; fish for TMAO).Only 1 MR study identified in this review explored dietary intake (lipidomic signatures of dairy consumption) in relation to a health outcome (blood pressure and cardiovascular disease risk) [73].Of note, the majority of the studies included combined cohorts with both men and pre-and postmenopausal women.Estrogen modifies the phosphatidylethanolamine-N-methyltransferase enzyme, creating differences in dietary choline requirements for men and postmenopausal women [82].Although included GWAS analyses adjusted for sex, the majority of MR analyses did not.Separating premenopausal women from men/postmenopausal women would likely identify sex-specific associations.In future analyses, participants should be analyzed according to sex and pre/postmenopausal status (separating premenopausal women from men/postmenopausal women), and hormone replacement therapy should be considered.
Lastly, our search strategy has a few limitations.Abstract and full-text screening may not have identified all relevant articles in which choline/choline metabolites are mentioned exclusively in supplemental files.Metabolome-wide analyses (particularly GWAS) have become more common, examining hundreds of circulating metabolite genome-wide associations.Authors report a small fraction of these associations within the main text, and publishing the full list of single nucleotide polymorphisms-xmetabolite associations would require the creation of a database.Lastly, ethnicity was largely European, which limits translation to other ethnicities.
In conclusion, there is a growing body of evidence illustrating the association of genetic polymorphisms with circulating choline and choline-related metabolite concentrations, as well as with numerous health outcomes.It is crucial to account for sex, menopausal status, and hormone replacement therapy in future analyses, specifically by separating premenopausal women and men/postmenopausal women.Future large GWAS and MR studies are needed that incorporate choline-related dietary intake into these types of analyses, as it may help to disentangle the varying consequences of genetic variants on circulating choline/choline metabolite concentrations and health outcomes.

TABLE 1
Eligibility criteria for inclusion in the review

Identification of studies via databases and registers Identification of studies via other methods
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TABLE 3
Choline/choline metabolites (n ¼ 16) reported to have a significant association with a health outcome in Mendelian randomization studies